1. Beta-Galactosidase Digest from 500 fmole Loaded on a 1-D Gel A. B. C. Bovine Serum Albumin Digest from 250 fmole Loaded on a 1-D Gel Automation of In-Gel Digestion for Protein Identification Libby Kellard¹, Aldo M. Pitt 1, Judi Tilghman², Jennifer Van Dinther 2, Sara Gutierrez¹, Claire Hapenney 3, and Marcy Engelstein¹ ¹Millipore Corporation, Danvers, MA 01923, ²PerkinElmer Life Sciences, Downers Grove, IL 60515, ³Applied Biosystems, Framingham MA 01701 Abstract The use of MALDI-TOF mass spectrometry for protein characterization and identification has dramatically improved the amount of information that can be obtained from biological samples and has increased the need for higher sample throughput. To address this need an automated approach for in-gel digestion and MALDI target spotting is presented. This poster describes the automation of the Millipore Montage In-Gel Digest 96 Kit using a Packard MultiPROBE ® II HT EX with Gripper Integration Platform for liquid handling and an Applied Biosystems Proteomics Solution 1™ System (PS1) for sample tracking and data analysis. All steps in the process are automated with the exception of excision of the protein spots/bands from polyacrylamide gels and the incubation of the protein with trypsin. Sample information for each gel slice is entered into the MulitPROBE at the start of the in-gel digestion procedure. Digested proteins are spotted on the MALDI target using Packard’s ABI MALDI spotting kit. After spotting the sample information is exported to the PS1 system for automated sample acquisition, data processing, database searching and report generation. The combination of Millipore’s kit and optimized protocol along with liquid handling from Packard and analysis from ABI provides a reproducible and automatable method for in-gel digestion and clean up of peptides prior to MALDI-TOF MS analysis. The successful automation of such complicated and lengthy procedures provides the throughput and simplicity necessary to meet the challenges of Proteomics. Proteomics Solution 1 System Advanced Result Browser In-Gel Digest 96 Kit Method Deck Layout on MultiPROBE II HT EX for Montage In-Gel Digest 96 Kit Summary Acknowledgments Richard Pintal from Applied Biosystems for the use of the Voyager-DE PRO. Copyright 2002 Millipore Corporation ABI MALDI Spotting Kit for the Packard MultiPROBE II Montage In-Gel Digest 96 Kit Optimized for Packard MultiPROBE and ABI Proteomics Solution I System Proteomics Solution 1 System Kit contains MultiScreen filter plates, Trypsin, and all the reagents for destaining, digestion, and extraction of 384 samples. The MultiScreen plate containing gel slices is placed on the vacuum manifold where destaining and extraction occur (after off-line incubation). Spotting digested proteins onto the ABI MALDI target takes place using the ABI MALDI Spotting Tower from Perkin Elmer. 1. Add 200 µl destain solution per well. 2. 20 minute incubation. 3. Filter to waste. 4. Repeat 3 times. 1. Add 50 µl of trypsin per well. 2. 37 o C Incubation occurs off- line (overnight). 1. Add 50 µl extraction solution per well. 2. Incubate for 30 minutes. 3. Filter peptide digestions and collect in v-bottom plate. 1. Spot 1 µl sample. 2. Overlay with 1 µl matrix. 1. Add 5 µl of 0.1% TFA to dried peptides. 2. Spot 0.5 µl of reconstituted sample. 3. Add 0.5 µl matrix to spotted sample. 1. Add 10 µl of 0.1% TFA to dried peptides. 2. Peptides bound to ZipTip. 3. Purified and concentrated peptides are eluted in 1.2 µl matrix onto the MALDI target. MultiPROBE WinPREP TM Software Digestion, extraction and spotting protocols are developed using MultiPROBE WinPREP automation control software. WinPREP allows for easy deck layout changes as well as control of vacuum and of the Gripper Integration Platform. Peptide digests are spotted onto an ABI MALDI target (96 x 2) using the ABI MALDI Spotting Kit from Perkin Elmer. Spotting of the peptides can be done using Packard’s spotting tips or Millipore’s Packard MultiPROBE II Compatible ZipTip µ-C18. ZipTip µ-C18 16% Coverage Reconstitute and spot 6% Coverage Gel bands were excised and processed using the Montage In-Gel Digest 96 Kit and the Packard MultiPROBE Liquid Handling System. Digested proteins were spotted utilizing 2 different methods. First the dried peptides were reconstituted and spotted directly (B). The same samples were then purified with ZipTip µ-C18 (A). MALDI analysis was done using ABI Voyager-DE TM PRO with PS1 software and Opti-Plate, the mass accuracy feature of Voyager 5.1 Software. Gel bands were excised and processed using the Montage In-Gel Digest 96 Kit and the Packard MultiPROBE Liquid Handling System. Digested proteins were spotted utilizing 2 different methods. First the dried peptides were reconstituted and spotted directly (B). The same samples were then purified with ZipTip µ-C18 (A). MALDI analysis was done using ABI Voyager-DE PRO with PS1 software and Opti-Plate, the mass accuracy feature of Voyager 5.1 Software Beta-Galactosidase Digest from 250 fmole Loaded on a 1-D Gel ZipTip µ-C18 23 % Coverage Reconstitute and Spot 13 % Coverage Gel bands were excised and processed using the Montage In-Gel Digest 96 Kit and the Packard MultiPROBE Liquid Handling System. Digested proteins were spotted utilizing 3 different methods. Peptides were spotted prior to the drying step (C). After drying the peptides were reconstituted and spotted directly (B). Finally, peptide digestions were purified with ZipTip µ-C18 (A). MALDI analysis was done using ABI Voyager-DE PRO with PS1 software and Opti-Plate, the mass accuracy feature of Voyager 5.1 Software. ZipTip µ-C18 27 % Coverage Reconstitute and Spot 18 % Coverage Direct Spot 9% Coverage Sample position and description information are exported from the MultiPROBE II system using sample tracking software. This information is imported into the PS1 system to create acquisition methods. The Proteomics Solution 1 Advanced Results Browser provides graphical analysis tools to compare MOWSE scores, percent protein coverage, and average mass accuracy for the complete set of samples on the 96 well MALDI sample plate. The Advanced Results Browser can generate batch reports which contains all the same information. Millipore’s Montage In-Gel Digest 96 Kit, Packard’s MultiPROBE II Liquid Handling System and ABI’s Proteomics Solution 1 System provide a total solution for automation of in-gel digestion and sample analysis. More abundant proteins (typically >500fmole) can be directly spotted after extraction to eliminate the dry down process. Reconstitution and spotting of samples is shown to be an acceptable method. In many cases the use of a ZipTip µ-C18 provided better results. PS1 software fully automates the data collection and query process for a significant time savings. Packard MultiPROBE II automates most steps in the In-Gel Digestion process. Excise Gel Pieces Destain Gel Pieces Digest Proteins Extract PeptidesDirect Spot Dry Peptides ZipTip and Spot Reconstitute and Spot MALDI Spotting Tower A. B. MultiScreen Plate with Gel Slices A. B.